Chromosomal multiple antibiotic resistance in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of about 40 chromosomal genes called the mar/sox/rob regulon. The major goals of this project are to understand the regulation of these activators, the mechanisms whereby they activate the regulon promoters, and the mechanisms whereby the multiple antibiotic resistance is generated.[unreadable] [unreadable] Upregulation of the transcriptional activators, MarA, SoxS and Rob can be effected by treating the cells with certain chemicals. Certain phenolic compounds derepress the marRAB operon; superoxides activate SoxR which in turn activates SoxS; and bile salts & other compounds activate the Rob protein directly. Thus, the upregulation of these activators can indicate the presence of such substances in the environment.[unreadable] [unreadable] We have found that certain mutations in E. coli upregulate all three activators. In particular, tolC mutants which do not have a functional TolC outer membrane channel have elevated transcription of marRAB & soxS and elevated activity of Rob protein. TolC is a vital component of several efflux pumps in E. coli & other enteric bacteria which play important roles in ridding bacteria of multiple antibiotics, bile salts, organic solvents and other xenobiotics.[unreadable] [unreadable] Using transcriptional fusions, we found that in tolC mutants marRAB and soxS transcription and Rob protein activity was upregulated by three distinct mechanisms: 1)The activation of marRAB transcription was independent of soxR and rob. 2) The activation of soxS transcription required SoxR. 3) Rob was post-transcriptionally activated. This is analogous to the regulation of these activators found upon treating cells with either phenolic compounds (marRAB), superoxides (soxS) or bile salts (Rob), respectively. The transcription of other regulon promoters, including tolC itself, was also elevated in tolC mutants. This upregulation is seen in cells that are growing in broth or in minimal media without any antibiotics or other xenobiotics. We attribute this upregulation to the intracellular accumulation of metabolic waste products that increase the transcription of marRAB and soxS and the post-transcriptional activation of Rob. This implies that TolC is normally involved in the efflux of cellular metabolites and not merely of xenobiotics.